Rapid-acting insulin analogues of enhanced stability

ABSTRACT

A two-chain insulin analogue contains a modified A-chain polypeptide and a modified B-chain polypeptide. The A-chain polypeptide comprises one or more of: a His or Glu substitution at position A8, a Glu substitution at position A14; and a Gln or Arg substitution at positon A17. The B-chain polypeptide comprises one or more of: a deletion of the amino acids at position B1, B1-B2, B1-B3, B30 or a combination thereof; an Ala or Glu substitution at position B2; a Glu substitution at position B3. The analogue exhibits thermodynamic stability in a zinc-free solution, decreased self-association, maintains biological potency, and no increased mitogenicity. The analogue exhibits resistance to chemical degradation and physical degradation. A method of treating a patient with diabetes mellitus or obesity comprises administering a physiologically effective amount of the insulin analogue or a physiologically acceptable salt thereof to a patient.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims benefit of pending U.S. Provisional ApplicationNo. 62/424,892 filed on Nov. 21, 2016.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under grant numbersDK040949 and DK074176 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

BACKGROUND OF THE INVENTION

This invention relates to polypeptide hormone analogues that exhibitenhanced pharmaceutical properties, such as increased thermodynamicstability, decreased mitogenicity, and feasibility of a rapid-actingformulation at high protein concentrations (1-5 mM) in the absence ofzinc ions. More particularly, this invention relates to insulinanalogues that confer rapid action at increased formulation strengths(relative to wild-type insulin) and/or that enable use of a broaderrange of excipients in a pharmaceutical formation (relative to wild-typeinsulin or conventional insulin analogues as traditionally formulated aszinc-ion-stabilized protein assemblies).

The engineering of non-standard proteins, including therapeutic agentsand vaccines, may have broad medical and societal benefits. Naturallyoccurring proteins—as encoded in the genomes of human beings, othermammals, vertebrate organisms, invertebrate organisms, or eukaryoticcells in general—may have evolved to function optimally within acellular context but may be suboptimal for therapeutic applications.Analogues of such proteins may exhibit improved biophysical,biochemical, or biological properties. A benefit of protein analogueswould be to achieve enhanced “on-target” activity (such as metabolicregulation of metabolism leading to reduction in blood-glucoseconcentration) with decreased unintended and unfavorable side effects,such as promotion of the growth of cancer cells. Another benefit of suchprotein engineering would be preservation of rapid onset of action onconcentration of the protein to achieve formulations of higher strength.Yet another example of a societal benefit would be enhancedcompatibility with a delivery device, such as an insulin pump or aclosed-loop system in which an algorithm connects the flow rate of thepump to the output of a continuous glucose monitor. An example of atherapeutic protein is provided by insulin. Wild-type human insulin andinsulin molecules encoded in the genomes of other mammals bind toinsulin receptors is multiple organs and diverse types of cells,irrespective of the receptor isoform generated by alternative modes ofRNA splicing or by alternative patterns of post-translationalglycosylation. Wild-type insulin also binds with lower but significantaffinity to the homologous Type 1 insulin-like growth factor receptor(IGF-1R).

Insulin is a two-chain protein molecule that in a vertebrate animal isthe biosynthetic product of a single-chain precursor, designatedproinsulin. The sequence and structure of human proinsulin areillustrated in FIGS. 1A (SEQ ID NO: 1) and 1B, respectively; thesequence of human insulin is shown in FIG. 1C. Insulin contains twopolypeptide chains, an A chain, containing 21 residues (SEQ ID NO: 2),and a B chain containing 30 residues(SEQ ID NO: 3). Specific amino acidsin one or the other chain are designated below by the standardthree-letter code (for example, “Ala” for Alanine or “Asp” for AsparticAcid) followed by a letter, optionally in superscript, that designatesthe chain (A or B) and position number in that chain relative to wildtype insulin. For example, Histidine at position 10 of the B chain isdesignated His^(B10), Valine at position 12 of the B chain is designatedVal^(B12), and Threonine at position 8 of the A chain is designatedThr^(A8). Alternatively, amino acids may be designated by the standardone-letter code (for example, “A” for Alanine or “D” for Aspartic Acid)followed by a letter designating the A or B chain, followed by positionnumber relative to wild type insulin. Under this convention, Histidineat position 10 of the B chain is designated HB10, Valine at position 12of the B chain is designated VB12, and Threonine at position 8 of the Achain is designated TAB. The numbering of the amino acids is maintainedrelative to wild type insulin, even in the presence of deletions ofamino acids from the N-terminal end of a polypeptide. Therefore, in ades B1 insulin analogue, which indicates that the first amino acid hasbeen deleted from the N-terminal end of the wild type sequence, thefirst amino acid of the B-chain is Valine, which is still designated asoccupying the B2 position. Likewise, in a des [31, B2] insulin analogue,in which the first two N-terminal amino acids have been deleted from awild type sequence, the first amino acid is Asparagine, which is stilldesignated as occupying the B3 position.

The term “insulin analogues” designates a class of molecules related towild-type insulin by substitution of one more amino-acid residues by adifferent type of amino acid or by modifications of one or more atoms inthe side chain or main chain of such residues by a different atom or setof atoms, but will still maintain at least a portion of one or moreproperties of wild-type insulin, such as the ability to bind insulinreceptor (IR). An example of an insulin analogue known in the art isinsulin lispro, in which Pro^(B28) is substituted by Lys and Lys^(B29)is substituted by Pro. Insulin lispro (also designated KP-insulin) isthe active component of the product Humalog® (Eli Lilly and Co.).

It is known in the art that the B chain of insulin may be modifiedthrough amino-acid substitutions at one or a few positions to enhancethe rate of absorption of an insulin analogue formulation from thesubcutaneous depot. Insulin formulations of increased strength(international units per ml) promise to be of particular benefit forpatients who exhibit marked insulin resistance and may also be of valuein internal or external insulin pumps, either to extend the reservoirlife or to permit miniaturization of the reservoir in a new generationof pump technologies.

Existing insulin products typically exhibit prolonged pharmacokineticand pharmacodynamics properties on increasing the concentration of theinsulin or insulin analogue to achieve formulation strengths greaterthan or equal to U-200 (200 international units/m1). Such prolongationimpairs the efficacy of such products for the prandial control ofglycemia on subcutaneous injection and impairs the efficacy and safetyof pump-based continuous subcutaneous infusion. In light of thesedisadvantages, the therapeutic and societal benefits of rapid-actinginsulin analogue formulations would be enhanced by the engineering ofinsulin analogues that retain rapid action at strengths between U-200and U-1000 (inclusive of these lower and upper bounds). Additionalbenefits would accrue if the novel soluble insulin analogue exhibitedweaker affinity for the Type 1 IGF receptor relative to wild-type humaninsulin. Still additional therapeutic and societal benefit would accrueif the concentrated insulin analogue formulation should exhibit reducedmitogenicity in assays developed to monitor insulin-stimulatedproliferation of human cancer cell lines and/or in assays developed tomonitor insulin-directed changes in gene expression associated with thestimulation or arrest of cellular proliferation.

Administration of insulin has long been established as a treatment fordiabetes mellitus. A major goal of conventional insulin replacementtherapy in patients with diabetes mellitus is tight control of the bloodglucose concentration to prevent its excursion above or below the normalrange characteristic of healthy human subjects. Excursions below thenormal range are associated with immediate adrenergic or neuroglycopenicsymptoms, which in severe episodes lead to convulsions, coma, and death.Excursions above the normal range are associated with increasedlong-term risk of microvascular disease, including retinopathy,blindness, and renal failure.

Insulin is a small globular protein that plays a central role inmetabolism in vertebrates. The hormone is stored in the pancreaticβ-cell as a Zn²⁺-stabilized hexamer, but functions as a Zn²⁺-freemonomer in the bloodstream. Insulin is the product of a single-chainprecursor, proinsulin, in which a connecting region (35 residues) linksthe C-terminal residue of B chain (residue B30) to the N-terminalresidue of the A chain. A variety of evidence indicates that it consistsof an insulin-like core and disordered connecting peptide. Formation ofthree specific disulfide bridges (A6-A11, A7-B7, and A20-B19) is coupledto oxidative folding of proinsulin in the rough endoplasmic reticulum(ER). Proinsulin is converted to insulin in the trans-Golgi network enroute to storage as zinc insulin hexamers in the glucose-regulatedsecretory granules within pancreatic beta-cells.

FIG. 2 is a schematic representation of the pharmacokinetic principlesunderlying the design of prandial (rapid-acting) insulin analogs asknown in the art. Whereas in a subcutaneous depot the insulin hexamer(upper left) is too large to efficiently penetrate into capillaries(bottom), more rapid uptake is mediated by the insulin dimer (centertop) and insulin monomer (upper right). Prandial insulin productsHumalog® and Novolog® contain insulin analogues (insulin lispro andaspart, respectively) with amino-acid substitutions at or near thedimerization surface of the zinc insulin hexamer such that its rate ofdisassembly is accelerated; prandial insulin product Apidra® (insulindeglulisine) is formulated as zinc-free oligomers (as a coupledequlibria) that likewise exhibit rapid rates of diassasembly in thedepot. The insulin analogues of the present invention provide isolatedinsulin monomers and weakly associating dimers whose augmented stabilityenables formulation in the absence of zinc-mediated assembly orzinc-free higher-order assembly. The hexamer at upper left depicts aT₃R^(f) ₃ zinc hexamer in which the A chain is shown in light gray, andB chain in dark gray; three bound phenolic ligands are shown as CPKmodels (paired ovals). The dimer at center depicts a zinc-free T2 dimerin which the A chain is shown in dark gray, and B chain in light(residues B1-B23 and B29-B30) or medium gray (B24-B28; anti-parallelbeta-sheet); dimer-related inter-chain hydrogen bonds are shown indotted line. A collection of crystallographic protomers (T, R and R^(f))is shown at upper right wherein the A chain is shown in dark gray, and Bchain in medium gray (B1-B9) and light gray (B10-B30).

Rapid action of insulin analogue formulations following theirsubcutaneous injection may reflect the pharmacokinetic properties ofprotein absorption from the subcutaneous depot and/or thepharmacodynamics properties of the insulin analogue once the insulinreceptor is engaged within target tissues Amino-acid substitutionswithin insulin analogue formulations of the present invention may employone or both of these mechanisms.

Substitutions at positions B28 and/or B29 are known in the art to impairthe strength of zinc-free insulin dimerization and the stability of thezinc insulin analogue hexamer. However, substitution of the wild-typeresidues Pro^(B28)-Lys^(B29) by Lys^(B28)-Pro^(B29) (insulin lispro, theactive component of Humalog® in a zinc-stabilized formulation) andsubstitution of Pro^(B28) by Asp (insulin aspart, the active componentof Novolog® in a zinc-stabilized formulation) impair the thermodynamicstability, chemical stability and physical stability of insulin,demonstrating that in general amino-acid substitutions in insulin confera tradeoff between favorable and unfavorable properties. Substitution ofLys^(B29) by Glu (one of two substitutions in insulin glulisine, theactive component of Apidra) with retention of wild-type Pro^(B28) has amore limited effect on dimerization or hexamer stability, but permitsstable formulation in a zinc-free solution at neutral pH. Despitepotential pharmacodynamics advantages of a foreshortened tail of insulinaction, such substitutions may incur tradeoffs in protein design as theintroduced amino-acid residues impair the stability or receptor-bindingaffinity of insulin to an extent that is difficult to predict.

There are medical and societal needs for a rapid-acting insulin analogueor even an ultra-rapid-acting insulin analogue in a soluble formulationat neutral pH at strengths in the range U-200 through U-1000 (inclusiveof these lower and upper values). A barrier to such products has longbeen posed by the complex self-association properties of wild-typeinsulin, which at neutral pH can form a concentration-dependentdistribution of monomeric, dimeric, trimeric, tetrameric, hexameric,dodecameric, and higher-order species. Traditional insulin formulationsknown in the art typically employ a predominance of zinc insulinhexamers at a nominal protein concentration, in monomer units, of 0.6 mM(lower on dilution), whose native self-assembly and stabilized by thezinc ions and protects the hormone from physical and chemicaldegradation. Concentrating wild-type insulin hexamers above this proteinconcentration leads to progressive hexamer-hexamer interactions; thisfurther level of self-association is associated with delayed absorptionof the injected insulin from a subcutaneous depot, leading in turn toprolonged pharmacokinetics and pharmacodynamics Analogous prolongationof current prandial insulin analogue products (Humalog®, Novolog® andApidra®) occurs on their concentration above ca. 2 mM (in monomerunits).

To overcome this barrier, we envisaged a novel route toward theengineering of rapid-acting insulin analogue formulations with increasedstrength. The first approach was to design ultra-stable insulin monomerswhose dimerization and higher-order self-assembly at proteinconcentrations as high as 3-8 mM would not impose kinetic barriers toprotein disassembly in the subcutaneous depot. In this approach theaugmented intrinsic stability of the individual insulin analoguemolecules would render its zinc-mediated hexamer assembly unnecessaryfor a stable formulation, i.e., in accordance with guidelines of theU.S. Food & Drug Administration with respect to chemical degradation,polymerization and fibrillation. In the absence of zinc coordination,the time scale of insulin self-assembly and disassembly is markedly morerapid than in the presence of zinc coordination; the difference isfurther magnified by the combination of zinc ions and preservativestypically employed in standard pharmaceutical formulations as thiscombination of excipients, which yields an “R₆” type hexamer that isexceptionally long-lived in the conformational equilibrium formed insuch a formulation. It is a feature of the present invention that suchanalogues retain at least 20% of the biological activity of wild-typeinsulin on a per-molecule basis.

An insulin analogue known in the art to exhibit enhanced intrinsicstability in the absence of zinc ions and decreased self-assembly beyondthe dimer is provided by Asp^(B10)-insulin. The wild-type residue(His^(B10)) functions in native hexamer assembly to coordinate the twoaxial zinc ions in the central axis of the hexamer. Substitution ofHis^(B10) by Asp impairs the binding of zinc ions in this axial mode andblocks higher-order self-assembly via the trimer-related surface of theclassical hexamer. Asp^(B10) may be expected on general grounds byenhance the segmental stability of the central B-chain α-helix in thezinc-free monomer or dimer via electrostatic mechanisms: as a favorableC-Cap residue and through potential formation of an (i, i+4) salt bridge(with Glu^(B14)). Irrespective of the theoretical underpinnings ofprotein stability, substitution of His^(B10) by Asp was observed indeedto augment the thermodynamic stability of the zinc-free insulin monomeras probed by chemical-denaturation studies. Asp^(B10) also enhances theaffinity of insulin for the insulin receptor and augments in parallelits potency to stimulate lipogenesis in isolated adipocytes. Despite theabove favorable properties conferred by substitution of His^(B10) by Aspin wild-type insulin, its clinical use was precluded by increasedmitogenicity in cell-culture assays of neoplastic cell lines (includinga cell line derived from a human breast cancer) in association with thefinding of an excess incidence of mammary tumors on chronic treatment ofSprague-Dawley rats by Asp^(B10)-insulin relative to wild-type insulin.The present invention excludes the use of Asp^(B10) as a stabilizingsubstitution due to its unfavorable association with carcinogenesis inSprague-Dawley rats. The present invention also excludes artificialamino acids known in the art to enhance the thermodynamic stability ofglobular proteins or non-polar peptide interfaces (such as fluorinatedaliphatic or aromatic side chains) as such unnatural amino acids areassociated with elevated manufacturing costs and unknown toxicity inmedical products intended for long-term use in patients or humansubjects.

SUMMARY OF THE INVENTION

It is, therefore, an aspect of the present invention to provide aninsulin analogue having enhanced thermodynamic stability in a zinc-freesolution while exhibiting decreased self-association, particularly athigher concentrations, such as those greater than 0.6 mM, and whilemaintinaning at least 20 percent of the biological potency of wild-typehuman insulin on a nanomolar basis. It is another aspect of the presentinvention that the insulin analogue does not exhibit enhancedmitogenicity. It is a further aspect that insulin analogue can bedesigned to exhibit sufficient zinc-free stability so as to conferresistance to chemical degradation and resistance to physicaldegradation—and therefore enable the development of rapid-actingpharmaceutical formulations on subcutaneous injection at strengthsU-100-U-300 and in some cases as high as U-500. It is still anotheraspect that such stability can be obtained in the absence of thecancer-associated Asp^(B10) substitution and in the absence ofartificial amino acids. The absence of the need for zinc ions and theincreased intrinsic stability of these analogues will permit a broadrange of excipients to be feasible, including excipients that enhancethe absorption of the injected insulin analogue from the subcutaneousdepot into the bloodstream. It is further envisioned that the presentcombination of stabilizing elements confers sufficient stability topermit the additional incorporation of substitutions at sites designedto foreshorten the duration of insulin signaling once the insulinreceptor is engaged. We envisage that the products of the presentinvention will disproportionately benefit patients treated withcontinuous subcutaneous infusion via insulin pumps and further mayenable the miniaturization of such pumps.

The analogues of the present invention thus consist of two polypeptidechains that contain a novel combination of amino-acid substitutions suchthat the analogues exhibit (i) enhanced thermodynamic stability in azinc-free solution, (ii) decreased self-association at proteinconcentrations greater than 0.6 mM, and (iii) biological potency atleast 20% of that of wild-type human insulin on a nanomolar basis in adiabetic rat. To these ends, the analogues of the present inventioncontain novel combinations of (i) stabilizing substitutions (atpositions A8, A14, and/or B29), (ii) a modification that augmentsphysical stability (N-terminal B-chain deletions of one, two or threeresidues), optionally with (iii) substitutions near the N terminus ofthe B chain and/or at position A21 that enhance chemical stability; andadditionally optionally (iv) substitutions at or adjoining the “Site2”-related surface of insulin (positions B13, B17, B18, Al2, A13, A14and/or A17) designed to foreshorten the duration of signaling once theinsulin receptor is engaged. The latter substitutions may be stabilizingor destabilizing such that the combination of substitutions andmodifications provides an analogue whose thermodynamic stability isgreater than that of WT insulin. The analogues of the present inventionmay optionally contain one- or two-residue extensions of the B chain(residues B31 and B32). Specifically excluded are unnatural amino acidsand the mitogenic substitution Asp^(B10).

It is envisioned that the present invention can provide a medicalbenefit in the form of optimization of the pharmacokinetic properties ofa soluble insulin analogue formulation such that rapid onset of actionis retained in formulations of strengths in the range U-200 throughU-1000, i.e., between twofold and tenfold higher than conventional U-100insulin products (in this nomenclature “U-X” designates X internal unitsper ml of solution or suspension).

We envisage that insulin analogues and formulations of the presentinvention will have utility in the treatment of diabetes mellitus. Itis, therefore, an aspect of the present invention that analoguescontaining a combination of amino-acid substitutions and/or N-terminalB-chain deletions, as set forth herein, that enable its stableformulation in a zinc-free solution at neutral pH in the proteinconcentration range 0.6-6.0 mM such that “rapid action” is maintainedfollowing subcutaneous injection in an animal model of diabetesmellitus—“rapid action” being defined by the reference pharmacodynamicsprofile of Humalog® or Apidra® at a protein concentration 0.6 mM (i.e.,as in a standard U-100 insulin product) on subcutaneous injection in thesame animal model. It is an additional aspect of the present inventionthat the analogues exhibit thermodynamic stabilities, chemicalstabilities and physical stabilities equal to or greater than that ofinsulin lispro or insulin glulisine (the active components of Humalog®or Apidra®, respectively) when dissolved in a solution at neutral pH inthe absence of zinc ions or other divalent metal ions. It is anotheraspect of the present invention that the analogues exhibitmitogenicities in a tissue-culture assay of a human breast-cancer cellline and also mediate transcriptional activation of thecell-proliferation-associated cyclin-D1 gene that in each case are equalto or less than that of insulin lispro (KP-insulin). It is yet anotheraspect of the present invention to provide a method of recombinantmanufacture of insulin analogs containing N-terminal deletion ofresidues B1, B1-B2 or B1-B3, optionally in conjunction withsubstitutions at neighboring positions to retard their chemicaldegradation.

In general, the present invention provide insulin analogues that eachcontain multiple modifications (but excluding substitutions at positionB10 and without incorporation of unnatural amino acids) that togetherconfer rapid action under a broad range of protein concentrations in therange 0.6-6 mM and that together are compatible with stablepharmaceutical formulation in the absence of zinc ions or other divalentmetal ions. Substitutions are located at one or more of the followingpositions: A8, A14, A17, B2, B3, B28 and/or B29. The analogs mayoptionally contain N-terminal deletion of the B chain (up to andincluding B3) and substitutions at or near the new N terminus tomitigate chemical degradation. The present invention thus pertains to anovel class of insulin analogues containing a combination ofmodifications that together provide the long-sought clinical advantagesnot conferred by any one of the constituent modifications. In anotherversion of the present invention residue B30 is absent. In yet anotherembodiment, the analogue of the present invention may contain Glycine,Alanine or Aspartic Acid at position A21.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a schematic representation of the sequence of humanproinsulin (SEQ ID NO: 1) including the A- and B-chains and theconnecting region shown with flanking dibasic cleavage sites (filledcircles) and C-peptide (open circles).

FIG. 1B is a structural model of proinsulin, consisting of aninsulin-like moiety and a disordered connecting peptide (dashed line).

FIG. 1C is a schematic representation of the sequence of human insulinindicating the position of residues B27 and B30 in the B-chain. The topchain is the insulin A-chain (SEQ ID NO: 2) and the bottom chain is theinsulin B-chain (SEQ ID NO: 3).

FIG. 2 is a schematic representation prandial (rapid-acting) insulinanalogs as known in the art, including the insulin hexamer (upper left),the insulin dimer (center top) and insulin monomer (upper right) with arepresentation of the ability of each of these forms to efficientlypenetrate into capillaries (bottom).

FIG. 3 is a bar graph showing the blood glucose drop (in mg/dL) permicrogram of insulin analogue for several embodiments of the insulinanalogue of the present invention and insulin lispro.

FIG. 4 is a bar graph providing a comparison of the AG_(u) values forwild type human insulin (HI); insulin lispro (lispro); AspB10-insulinlispro (DB10, KP); and EAB, EA14, RA17 des B1, des B30 insulin (T-0688).

FIG. 5 is a bar graph providing the change in percentage of highmolecular weight protein (HMWP) of embodiments of the insulin analogueof the present invention at 45° C. for 7 days as determined byreverse-phase HPLC. All experimental samples were formulated withoutzinc.

FIG. 6 is a bar graph providing the fibrillation lag time of embodimentsof the insulin analogue of the present invention and that of insulinlispro in phosphate buffered saline (PBS), pH 7.4 at 40° C. with aconstant shake of 1000 cpm. All experimental samples were formulatedwithout zinc.

FIG. 7 is a bar graph providing the binding affinity of embodiments ofthe insulin analogue of the present invention to human type 1insulin-like growth factor receptor (hIGFR) relative to insulin lispro.Relative affinity is defined as the ratio of dissociation constants asdetermined by competitive displacement of bound ¹²⁵I-labeled humaninsulin. All experimental samples were formulated without zinc.

FIG. 8 is a bar graph providing the change in Cyclin D1 mRNAaccumulation utilizing a rat myoblast cell line (L6) with highexpression of insulin receptor as the cell model. All experimentalsamples were formulated without zinc.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed toward an insulin analogue thatprovides enhanced in vivo biological potency on a per-molecular basis,rapid action under a broad range of protein concentrations andformulation strengths (typically from U-100 to U-500, and optionally ashigh as U-1000), IR-A/IR-B receptor-binding affinities with absoluteaffinities in the range 5-100% relative to the affinities of wild-typehuman (the lower limit chosen to correspond to proinsulin), affinity forthe IGF-1R no greater than that of insulin lispro, and thermodynamicstability in the absence of zinc ions that is equal to or greater thanthat of human insulin lispro in the absence of zinc ions.

The above combination of features is conferred by a novel combination ofsubstitutions within the A- and B chains, optionally with N-terminaldeletion of the B chain, and optionally des-Thr^(B30). The A- and Bchain substitutions fall into five classes: (i) substitutions atSite-related positions (B13, B17, B18, A12, A13, A14 and/or A17); (ii)non-beta-branched substitutions at position A8; (iii) helicogenicsubstitutions at position A14 containing side chains that are eitherpolar, charged or smaller than the native Tyrosine; (iv) substitutionsat positions B28 and/or B29 as known in the art to decrease dimerizationof insulin or to enhance its solubility at neutral pH; and (v)substitutions near the N terminus of the B chain in conjunction withN-terminal deletions. Some of these substitutions may in isolationaugment the stability of wild-type insulin whereas others may inisolation impair the stability of wild-type insulin. Likewise, some ofthese substitutions may in isolation extend the tail of insulin action(on intravenous bolus injection) whereas others may in isolationmitigate or foreshorten this tail. An aspect of the invention provides acombination of such substitutions, optionally in conjunction withN-terminal deletion of the B chain, that together provide an insulinanalogue whose formulation under a broad range of protein concentrationsin the range 0.6-6.0 mM retains rapid action on subcutaneous injectionand exhibits adequate physical- and chemical stability to be practicalfor the treatment of diabetes mellitus.

Optimization of the C-CAP Residue of the A1-A8 α-Helix. The β-branchedside chain of Threonine at position A8 violates peptide-based rules ofα-helical propensity and C-CAP potential. Thr^(A8) is near but notwithin the classical receptor-binding surface of insulin as defined inco-crystal structures of insulin bound to fragments of the receptorectodomain. Non-β-branched substitutions at this position are known inthe art to stabilize the insulin molecule in the context of wild-typeinsulin and insulin lispro, but their functional compatibility andbiophysical effects on insulin analogues containing Site-2-relatedsubstitutions are unknown and difficult to predict because effects ofC-CAP substitutions in globular proteins are in general contextdependent.

Optimization of the Surface of the A12-A18 α-Helix. An unusual featureof the structure of the insulin monomer is the hyper-exposure ofTyrosine at position A14 on the surface of the A12-A18 α-helix. Theconformation of this side chain is variable among crystal structures andexhibits motional narrowing in ¹H-NMR studies under a variety ofsolution conditions. Substitution of such an exposed large non-polarside chain by a side chain of similar helical propensity that is smaller(such as Alanine), polar (such as Glutamine), or charged (such asGlutamic Acid or Arginine) might enhance overall stability by mitigatingthe reverse hydrophobic effect, but like C-CAP potential above, thiseffect is context-dependent and highly variable in magnitude. Too fewexamples of reverse hydrophobic effects have been described to enableeffects of A14 substitutions on the stability of insulin to bepredictable.

The N-terminal three residues of the B chain exhibit variable ordisordered conformations. Although deletion of residues B1, B1-B2 orB1-B3 of the B chain of insulin is known in the art not to impair theactivity of insulin or its binding of the hormone to the insulinreceptor, this “arm” contributes to the nascent folding efficiency ofproinsulin within mammalian cells. Whereas such deletions do not alterthe thermodynamic stability of the mature zinc-free hormone in solution,such deletions eliminate the solvent exposure of non-polar side chains(Phenylalanine at position B1 and Valine at position B2) and so mayreduce the tendency of the protein to undergo non-native aggregation,extending physical stability. Because such deletions may enhance thechemical degradation of residues at or near the new N terminus, deletionof residues B1, B1-B2 or B1-B3 may optionally be combined withneighboring amino-acid substitutions intended to mitigate such chemicaldegradation in a neutral-pH solution. Also without wishing to beconstrained by theory, we further envision that additive or non-additiveeffects of Site-2-related substitutions and Glu^(B29) attenuatemitogenic signaling by such analogues on binding to the insulin receptorand would be associated with reduced binding to the mitogenic Type 1 IGFreceptor.

It is an aspect of the present invention that rapid absorption kineticsfrom a subcutaneous depot may be generated by an insulin analogue thatis monomeric, dimeric, trimeric, tetrameric or hexameric—but not is ahigher-order state of self-assembly—in a zinc-free solution at neutralpH at a protein concentration of 0.6-6.0 mM (as calculated in relationto the formal monomer concentration). In the absence of zinc ions orother divalent metal ions, such native self-association is characterizedby rapid exchange among states of self-assembly (i.e., with rateconstants of dissociation such that lifetimes of the component dimers,trimers, tetramers and hexamers, to the extent that they are present inthe equilibrium, are less than 1 second). Such rapid exchange stands incontrast to lifetimes of hours observed among zinc-stabilized insulinhexamers. Conventional prandial products, as known in the art, representa continuum of possible coupled equilibria between states ofself-assembly, including zinc-stabilized or zinc-ion-independenthexamers extended by potential hexamer-hexamer interactions. Molecularimplementation of this strategy provides a novel class of insulinanalogues that (i) are as stable or more stable as a zinc-free monomerand dimer relative to insulin lispro and (ii) retain at least a portionof the biological potency of wild-type human insulin (as assessed byhormone-regulated reduction in blood-glucose concentration) on aper-molecular or per-nanomole basis. It is a feature of the presentinvention that retained potency in relation to glycemic control isassociated with a mitogenicity that is no higher than that of insulinlispro. For many of the analogues of the present invention, mitogenicityis reduced relative to wild-type insulin, a reduction that is abiological consequence of a distinct signaling pathway that isundesirable from the perspective of cancer risk and cancer growth.

It is also envisioned that insulin analogues may be made with A- and Bchain sequences derived from animal insulins, such as porcine, bovine,equine, and canine insulins, by way of non-limiting examples, so long asno substitutions are present at position B10 and no unnatural aminoacids are utilized. Such variant B chains derived from human insulin oranimal insulins may optionally lack Thr^(B30) (des-B30) or contain aC-terminal dipeptide extension (with respective residue positionsdesignated B31 and B32) wherein at least one of these C-terminalextended residues is an acidic amino acid. In addition or in thealternative, the insulin analogue of the present invention may contain adeletion of residues B 1, B 1-B2, or B 1-B3; or may be combined with avariant B chain lacking Proline at position B28 (e.g., LyS^(B28),Ala^(B28) or Gln^(B28) in combination with Glutamic Acid at positionB29).

It is further envisioned that the insulin analogues of the presentinvention may be derived from Lys-directed proteolysis of a precursorpolypeptide in yeast biosynthesis in Pichia pastoris, Saccharomycescerevisciae, or other yeast expression species or strains. Such strainsmay be engineered to encode a Lysine at positions B1, B2 or B3 in orderto enable post-fermentation processing of a precursor containing anN-terminal B-chain extension such that analogues respectively lackingresidues B 1, B 1-B2 or B 1-B3 are producted. The new N-terminal residuemay optionally be substituted by Glutamic Acid or Alanine. The variant Bchain of a des-B 1 analogue may therefore begin with an N-terminalValine (the native B2 residue in wild-type insulin), Alanine or GlutamicAcid and optionally Alanine or Glutamic Acid at the second position; thevariant B chain of a des-[B1-B2] analogue may likewise begin with anN-terminal Asparagine (the native B2 residue in wild-type insulin),Alanine or Glutamic Acid at the first position and optionally Alanine orGlutamic Acid at the second position; the variant B chain of ades-[B1-B3] analogue may likewise begin with an N-terminal Glutamine(the native B3 residue in wild-type insulin), Alanine or Glutamic Acid.Such substitutions at or adjoining the new N terminus of variant B chainare intended to avoid chemical degradation of Asparagine at wild-typeposition B3 and Glutamine at wild-type position B4. Such substitution ofGlutamic Acid would also augment the net negative charge of the insulinanalogue at neutral pH, which would be expected to enhance itssolubility at neutral pH.

Furthermore, in view of the similarity between human and animalinsulins, and use in the past of animal insulins in human patients withdiabetes mellitus, it is also envisioned that other minor modificationsin the sequence of insulin may be introduced, especially thosesubstitutions considered “conservative.” For example, additionalsubstitutions of amino acids may be made within groups of amino acidswith similar side chains, without departing from the present invention.These include the neutral hydrophobic amino acids: Alanine (Ala or A),Valine (Val or V), Leucine (Leu or L), Isoleucine (Ile or I), Proline(Pro or P), Tryptophan (Trp or W), Phenylalanine (Phe or F) andMethionine (Met or M). Likewise, the neutral polar amino acids may besubstituted for each other within their group of Glycine (Gly or G),Serine(Ser or S), Threonine (Thr or T), Tyrosine (Tyr or Y), Cysteine(Cys or C), Glutamine (Glu or Q), and Asparagine (Asn or N). Acidicamino acids are Aspartic acid (Asp or D) and Glutamic acid (Glu or E).Introduction of basic amino-acid substitutions (including Lysine (Lys orK), Arginine (Arg or R) and Histidine (His or H)) are not preferred inorder to maintain the enhanced net negative charge of this class ofanalogues. Unless noted otherwise or wherever obvious from the context,the amino acids noted herein should be considered to be L-amino acids.Standard amino acids may also be substituted by non-standard amino acidsbelonging to the same chemical class.

The amino-acid sequence of human proinsulin is provided, for comparativepurposes, as SEQ ID NO: 1.

(human proinsulin) SEQ ID NO: 1Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe-Tyr-Thr-Pro-Lys-Thr-Arg-Arg-Glu-Ala-Glu-Asp-Leu-Gln-Val-Gly-Gln-Val-Glu-Leu-Gly-Gly-Gly-Pro-Gly-Ala-Gly-Ser-Leu-Gln-Pro-Leu-Ala-Leu-Glu-Gly-Ser-Leu-Gln-Lys-Arg-Gly-Ile-Val-Glu-Gln-Cys-Cys-Thr-Ser-Ile-Cys-Ser-Leu-Tyr-Gln-Leu-Glu-Asn-Tyr- Cys-Asn

The amino-acid sequence of the A chain of human insulin is provided asSEQ ID NO: 2.

(human A chain; residue positions A1-A21) SEQ ID NO: 2Gly-Ile-Val-Glu-Gln-Cys-Cys-Thr-Ser-Ile-Cys-Ser-Leu-Tyr-Gln-Leu-Glu-Asn-Tyr-Cys-Asn

The amino-acid sequence of the B chain of human insulin is provided asSEQ ID NO: 3.

(human B chain; residue positions B1-B30) SEQ ID NO: 3Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe- Phe-Tyr-Thr-Pro-Lys-Thr

The amino-acid sequence of a modified insulin of the present inventionis given in general form in SEQ ID NO 4 and SEQ ID NO: 5 wherein the sixCysteine residues are paired to provide three disulfide bridges as inwild-type human insulin.

A chain SEQ ID NO: 4 Gly-Ile-Val-Glu-Gln-Cys-Cys-Xaa₁-Ser-Ile-Cys-Xaa₂-Xaa₃-Xaa₄-Gln-Leu-Xaa₅-Asn-Tyr-Cys-Xaa₆ B chain SEQ ID NO: 5Xaa₇-Xaa₈-Xaa₉-Xaa₁₀-His-Leu-Cys-Gly-Ser-His-Leu-Ala-Xaa₁₁-Ala-Leu-Tyr-Xaa₁₂-Xaa₁₃-Cys-Gly-Glu-Arg-Gly-Phe-Phe-Tyr-Thr-Xaa₁₄-Xaa₁₅-Thr-Xaa₁₆-Xaa₁₇

Where Xaa₁ (position A8) may be Thr (as in wild-type insulin), His, Gluor any other non-β-branched amino acid; where Xaa₂ may be Ser (as inwild-type insulin), Ala, Asp, Glu or His; where Xaa₃ may be Leu (as inwild-type insulin), Ala, Glu, His, Phe, Tyr or Trp; where Xaa₄ may beTyr (as in wild-type insulin), Ala, Glu, Gln, His or Leu; where Xaa₅ maybe Glu (as in wild-type insulin), Ala, Arg, Gln, His, Leu, Phe or Tyr;where Xaa₆ may be Asn (as in wild-type insulin), Ala, Gly or Glu; whereXaa₇-Xaa₈-Xaa₉ may be Phe-Val-Asn as in wild-type human insulin orN-terminal deleted variants Val-Asn (des-B1; no residue Xaa₇), Asn(des-B1, B2; no residue Xaa₇ and no residue Xaa₈) or omitted (des-B1-B3;no residue Xaa₇, no residue Xaa₈ and no residue Xaa₉), or optionally arespective des-B1 variant (beginning Ala-Asn, Glu-Asn, Ala-Ala, Ala-Gluor Glu-Glu), a des-B1, B2 variant (beginning Asn-Glu, Asn-Ala, Ala-Asn,Glu-Asn, Ala-Ala, Ala-Glu, Glu-Ala or Glu-Glu), or a des-B1-B3 variant(beginning Ala-His or Glu-His); where Xaa₁₀ may be Gln (as in wild-typeinsulin), Ala or Glu; where Xaa₁₁ may be Glu (as in wild-type insulin),Ala, Gln, His or Leu; where Xaa₁₂ may be Leu (as in wild-type insulin),Ala, Arg, Glu, His, Lys, Phe, Trp, Tyr or Val; where Xaa₁₃ may be Val(as in wild-type insulin), Ala, His, Leu, Lys, Glu, Phe, Thr, Trp orTyr; where Xaa₁₄ may be Pro (as in wild-type insulin), Ala, Arg, Glu, orLys; where Xaa₁₅ may be Lys (as in wild-type insulin), Ala, Arg, Glu, orPro; and where optionally Xaa₁₆-Xaa₁₇ provides a C-terminal monopeptideor dipeptide extension of the B chain such that at least one residue isan acidic side chain (Asp or Glu). Analogues of the present inventionmay lack C-terminal extention Xaa₁₆-Xaa₁₇ and also lack Thr^(B30) (orany other amino acid) at position B30.

Thus, analogues of the present invention may optionally containN-terminal deletions of the B chain (des-B1, des-B1, B2 or des-B1-B3) asindicated in SEQ ID NOs: 6-26. These N-terminal residues are notrequired for receptor binding, but their presence in a biosyntheticsingle-chain precursor is thought to enhance the efficiency of nativedisulfide pairing in the endoplasmic reticulum and thus productionyields. By way of non-limiting examples, the following sequencesexemplify variant B chains containing N-terminal deletions.

Examples of specific A-chain and B-chain sequences according to thepresent invention include SEQ ID NOs: 6-104.

SEQ ID NO 6: des-(B1) derivative of Glu^(B28)-insulinVal-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe- Tyr-Thr-Glu-Lys-ThrSEQ ID NO 7: des-(B1, B2) derivative of Glu^(B28)-insulinAsn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe-Tyr- Thr-Glu-Lys-ThrSEQ ID NO 8: des-(B1-B3) derivative of Glu^(B28)-insulinGln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe-Tyr-Thr- Glu-Lys-ThrSEQ ID NO 9: des-(B1) derivative of Asp^(B28)-insulinVal-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe- Tyr-Thr-Asp-Lys-ThrSEQ ID NO 10: des-(B1, B2) derivative of Asp^(B28)-insulinAsn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe-Tyr- Thr-Asp-Lys-ThrSEQ ID NO 11: des-(B1-B3) derivative of Asp^(B28)-insulinGln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe-Tyr-Thr- Asp-Lys-ThrSEQ ID NO 12: des-(B1) derivative of (Lys^(B28), Pro^(B29))-insulinVal-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe- Tyr-Thr-Lys-Pro-ThrSEQ ID NO 13: des-(B1, B2) derivative of (Lys^(B28), Pro^(B29))-insulinAsn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe-Tyr- Thr-Lys-Pro-ThrSEQ ID NO 14: des-(B1-B3) derivative of (Lys^(B28), Pro^(B29))-insulinGln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe-Tyr-Thr- Lys-Pro-ThrSEQ ID NO 15: des-(B1) derivative of (Ala^(B28))-insulinVal-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe- Tyr-Thr-Ala-Lys-ThrSEQ ID NO 16: des-(B1, B2) derivative of (Ala^(B28))-insulinAsn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe-Tyr- Thr-Ala-Lys-ThrSEQ ID NO 17: des-(B1-B3) derivative of (Ala^(B28))-insulinGln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe-Tyr-Thr- Ala-Lys-ThrSEQ ID NO 18: des-(B1) derivative of (Glu^(B28), Pro^(B29))-insulinVal-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe- Tyr-Thr-Glu-Pro-ThrSEQ ID NO 19: des-(B1, B2) derivative of (Glu^(B28), Pro^(B29))-insulinAsn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe-Tyr- Thr-Glu-Pro-ThrSEQ ID NO 20: des-(B1-B3) derivative of (Glu^(B28), Pro^(B29))-insulinGln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe-Tyr-Thr- Glu-Pro-ThrSEQ ID NO 21: des-(B1) derivative of (Ala^(B28), Pro^(B29))-insulinVal-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe- Tyr-Thr-Ala-Pro-ThrSEQ ID NO 22: des-(B1, B2) derivative of (Ala^(B28), Pro^(B29))-insulinAsn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe-Tyr- Thr-Ala-Pro-ThrSEQ ID NO 23: des-(B1-B3) derivative of (Ala^(B28), Pro^(B29))-insulinGln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe-Tyr-Thr- Ala-Pro-Thr

The insulin analogues of the present invention containing variant Bchains with the above N-terminal deletions may also contain amino-acidsubstitutions at or near the new N terminus as exemplified in thefollowing sequences as non-limiting examples.

SEQ ID NO 24: des-(B1) derivative of Glu^(B28)-insulin in whichthe variant B chain begins Ala-Asn, Glu-Asn,Val-Ala, Val-Glu, Glu-Ala, or Glu-Glu.Xaa-Xaa-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe-Tyr-Thr-Glu-Lys-Thr SEQ ID NO 25:des-(B1, B2) derivative of Glu^(B28)-insulin inwhich the variant B chain begins Asn-Glu,Asn-Ala, Ala-Gln, Glu-Gln, Ala-Glu, Glu-Glu, or Glu-Ala.Xaa-Xaa-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe- Phe-Tyr-Thr-Glu-Lys-ThrSEQ ID NO 26: des-(B1-B3) derivative of Glu^(B28)-insulin inwhich the new N-terminal residue is Ala or Glu.Xaa-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe-Tyr- Thr-Glu-Lys-ThrSEQ ID NO 27: des-(B1) derivative of Asp^(B28)-insulin in whichthe variant B chain begins Ala-Asn, Glu-Asn,Val-Ala, Val-Glu, Glu-Ala, or Glu-Glu.Xaa-Xaa-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe-Tyr-Thr-Asp-Lys-Thr SEQ ID NO 28:des-(B1, B2) derivative of Asp^(B28)-insulin inwhich the variant B chain begins Asn-Glu,Asn-Ala, Ala-Gln, Glu-Gln, Ala-Glu, Glu-Glu, or Glu-Ala.Xaa-Xaa-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe- Phe-Tyr-Thr-Asp-Lys-ThrSEQ ID NO 29: des-(B1-B3) derivative of Asp^(B28)-insulin inwhich the new N-terminal residue is Ala or Glu.Xaa-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe-Tyr- Thr-Asp-Lys-ThrSEQ ID NO 30: des-(B1) derivative of (Lys^(B28), Pro^(B29))-insulinin which the variant B chain begins Ala-Asn,Glu-Asn, Val-Ala, Val-Glu, Glu-Ala, or Glu-Glu.Xaa-Xaa-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe-Tyr-Thr-Lys-Pro-Thr SEQ ID NO 31:des-(B1, B2) derivative of (Lys^(B28), Pro^(B29))-insulin in which the variant B chain beginsAsn-Glu, Asn-Ala, Ala-Gln, Glu-Gln, Ala-Glu, Glu-Glu, or Glu-Ala.Xaa-Xaa-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe- Phe-Tyr-Thr-Lys-Pro-ThrSEQ ID NO 32: des-(B1-B3) derivative of (Lys^(B28), Pro^(B29))-insulin in which the new N-terminal residue is Ala or Glu.Xaa-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe-Tyr- Thr-Lys-Pro-ThrSEQ ID NO 33: des-(B1) derivative of (Glu^(B28))-insulin in whichthe variant B chain begins Ala-Asn, Glu-Asn,Val-Ala, Val-Glu, Glu-Ala, or Glu-Glu.Xaa-Xaa-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe-Tyr-Thr-Glu-Lys-Thr SEQ ID NO 34:des-(B1, B2) derivative of (Ala^(B28))-insulin inwhich the variant B chain begins Asn-Glu,Asn-Ala, Ala-Gln, Glu-Gln, Ala-Glu, Glu-Glu, or Glu-Ala.Xaa-Xaa-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe- Phe-Tyr-Thr-Ala-Lys-ThrSEQ ID NO 35: des-(B1-B3) derivative of (Ala^(B28))-insulin inwhich the new N-terminal residue is Ala or Glu.Xaa-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe-Tyr- Thr-Ala-Lys-ThrSEQ ID NO 36: des-(B1) derivative of (Glu^(B28), Pro^(B29))-insulinin which the variant B chain begins Ala-Asn,Glu-Asn, Val-Ala, Val-Glu, Glu-Ala, or Glu-Glu.Xaa-Xaa-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe-Tyr-Thr-Glu-Pro-Thr SEQ ID NO 37:des-(B1, B2) derivative of (Glu^(B28), Pro^(B29))-insulin in which the variant B chain beginsAsn-Glu, Asn-Ala, Ala-Gln, Glu-Gln, Ala-Glu, Glu-Glu, or Glu-Ala.Xaa-Xaa-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe- Phe-Tyr-Thr-Glu-Pro-ThrSEQ ID NO 38: des-(B1-B3) derivative of (Glu^(B28), Pro^(B29))-insulin in which the new N-terminal residue is Ala or Glu.Xaa-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe-Tyr- Thr-Glu-Pro-ThrSEQ ID NO 39: des-(B1) derivative of (Ala^(B28), Pro^(B29))-insulinin which the variant B chain begins Ala-Asn,Glu-Asn, Val-Ala, Val-Glu, Glu-Ala, or Glu-Glu.Xaa-Xaa-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe-Tyr-Thr-Ala-Pro-Thr SEQ ID NO 40:des-(B1, B2) derivative of (Ala^(B28), Pro^(B29))-insulin in which the variant B chain beginsAsn-Glu, Asn-Ala, Ala-Gln, Glu-Gln, Ala-Glu, Glu-Glu, or Glu-Ala.Xaa-Xaa-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe- Phe-Tyr-Thr-Ala-Pro-ThrSEQ ID NO 41: des-(B1-B3) derivative of (Ala^(B28), Pro^(B29))-insulin in which the new N-terminal residue is Ala or Glu.Xaa-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe-Tyr- Thr-Ala-Pro-Thr

Still further examples of A-chain and B-chain polpeptides according tothe present invention include the following:

SEQ ID NO: 42 (HA8 EA14 QA17):Gly-Ile-Val-Glu-Gln-Cys-Cys-His-Ser-Ile-Cys-Ser-Leu-Glu-Gln-Leu-Gln-Asn-Tyr-Cys-Asn SEQ ID NO: 43 (des B1 AB2 EB29):Ala-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe- Tyr-Thr-Pro-Glu-ThrSEQ ID NO: 44 (EA8 EA14 QA17):Gly-Ile-Val-Glu-Gln-Cys-Cys-Glu-Ser-Ile-Cys-Ser-Leu-Glu-Gln-Leu-Gln-Asn-Tyr-Cys-Asn SEQ ID NO: 45 (des B1 AB2 des B30):Ala-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe- Tyr-Thr-Pro-LysSEQ ID NO: 46 (des B1 EB2 des B30):Glu-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe- Tyr-Thr-Pro-LysSEQ ID NO: 47 (des [B1 B2] EB3 des B30):Glu-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe-Tyr- Thr-Pro-LysSEQ ID NO: 48 (des B1 des B30):Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe- Tyr-Thr-Pro-LysSEQ ID NO: 49 (HA8, EA14)Gly-Ile-Val-Glu-Gln-Cys-Cys-His-Ser-Ile-Cys-Ser-Leu-Glu-Gln-Leu-Glu-Asn-Tyr-Cys-Asn SEQ ID NO: 50 (des B1, EB29)Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe- Tyr-Thr-Pro-Glu-ThrSEQ ID NO: 51 (des B1 QB13 des B30)Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Gln-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe- Tyr-Thr-Pro-LysSEQ ID NO: 52 (EA8, EA14 RA17)Gly-Ile-Val-Glu-Gln-Cys-Cys-Glu-Ser-Ile-Cys-Ser-Leu-Glu-Gln-Leu-Arg-Asn-Tyr-Cys-AsnSEQ ID NO 53 (des B1 QB13 EB 22 des B30)Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Gln-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Glu-Gly-Phe-Phe- Tyr-Thr-Pro-LysSEQ ID NO: 54 (HA8 EA14 RA17)Gly-Ile-Val-Glu-Gln-Cys-Cys-His-Ser-Ile-Cys-Ser-Leu-Glu-Gln-Leu-Arg-Asn-Tyr-Cys-AsnSEQ ID NO: 55 (des B1 EB 22 des B30):Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Glu-Gly-Phe-Phe- Tyr-Thr-Pro-LysSEQ ID NO: 56 (des B1, QB13 EB29)Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Gln-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe- Tyr-Thr-Pro-Glu-ThrSEQ ID NO: 57 (HA8 WA13)Gly-Ile-Val-Glu-Gln-Cys-Cys-His-Ser-Ile-Cys-Ser-Trp-Tyr-Gln-Leu-Glu-Asn-Tyr-Cys-Asn SEQ ID NO: 58 (HA8 LA14 QA17):Gly-Ile-Val-Glu-Gln-Cys-Cys-His-Ser-Ile-Cys-Ser-Leu-Leu-Gln-Leu-Gln-Asn-Tyr-Cys-Asn SEQ ID NO: 59 (EA8 WA13)Gly-Ile-Val-Glu-Gln-Cys-Cys-Glu-Ser-Ile-Cys-Ser-Trp-Tyr-Gln-Leu-Glu-Asn-Tyr-Cys-Asn SEQ ID NO: 60 (EA8)Gly-Ile-Val-Glu-Gln-Cys-Cys-Glu-Ser-Ile-Cys-Ser-Leu-Tyr-Gln-Leu-Glu-Asn-Tyr-Cys-Asn SEQ ID NO: 61 (EA8 LA14 RA17):Gly-Ile-Val-Glu-Gln-Cys-Cys-Glu-Ser-Ile-Cys-Ser-Leu-Leu-Gln-Leu-Arg-Asn-Tyr-Cys-Asn SEQ ID NO: 62 (des B1 EB2 EB29):Glu-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe- Tyr-Thr-Pro-Glu-ThrSEQ ID NO: 63 (des B1 EB2 EB17 des B30):Glu-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Glu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe- Tyr-Thr-Pro-LysSEQ ID NO: 64 (des B1 EB2 FB17 des B30):Glu-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Phe-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe- Tyr-Thr-Pro-LysSEQ ID NO: 65 (des B1 EB2 NB17 des B30):Glu-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Asn-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe- Tyr-Thr-Pro-LysSEQ ID NO: 66 (des B1 AB2 EB17 des B30):Ala-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Glu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe- Tyr-Thr-Pro-LysSEQ ID NO: 67 (des B1 AB2 FB17 des B30):Ala-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Phe-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe- Tyr-Thr-Pro-LysSEQ ID NO: 68 (des [B1, B2] EB29)Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe-Tyr- Thr-Pro-Glu-ThrSEQ ID NO: 69 (des [B1, B2] des B30):Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe-Tyr- Thr-Pro-LysSEQ ID NO: 70 (des [B1, B2] QB13 des B30):Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Gln-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe-Tyr- Thr-Pro-LysSEQ ID NO: 71 (des [B1, B2], QB13 EB29)Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Gln-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe-Tyr- Thr-Pro-Glu-ThrSEQ ID NO: 72 (des [B1, B2] QB13 EB22 des B30):Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Gln-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Glu-Gly-Phe-Phe-Tyr- Thr-Pro-LysSEQ ID NO: 73 (des [B1, B2] EB 22 des B30):Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Glu-Gly-Phe-Phe-Tyr- Thr-Pro-LysSEQ ID NO: 74 (EA8 LA14 QA17):Gly-Ile-Val-Glu-Gln-Cys-Cys-Glu-Ser-Ile-Cys-Ser-Leu-Leu-Gln-Leu-Gln-Asn-Tyr-Cys-Asn SEQ ID NO: 75 (HA8)Gly-Ile-Val-Glu-Gln-Cys-Cys-His-Ser-Ile-Cys-Ser-Leu-Tyr-Gln-Leu-Glu-Asn-Tyr-Cys-Asn SEQ ID NO: 76 (EA8 LA14)Gly-Ile-Val-Glu-Gln-Cys-Cys-Glu-Ser-Ile-Cys-Ser-Leu-Leu-Gln-Leu-Glu-Asn-Tyr-Cys-Asn SEQ ID NO: 77 (EA8 EA14)Gly-Ile-Val-Glu-Gln-Cys-Cys-Glu-Ser-Ile-Cys-Ser-Leu-Glu-Gln-Leu-Glu-Asn-Tyr-Cys-Asn SEQ ID NO: 78 (EA8 QA17)Gly-Ile-Val-Glu-Gln-Cys-Cys-Glu-Ser-Ile-Cys-Ser-Leu-Tyr-Gln-Leu-Gln-Asn-Tyr-Cys-Asn SEQ ID NO: 79 (EA8 WA13 EA14)Gly-Ile-Val-Glu-Gln-Cys-Cys-Glu-Ser-Ile-Cys-Ser-Trp-Glu-Gln-Leu-Glu-Asn-Tyr-Cys-Asn SEQ ID NO: 80 (HA8 WA13 EA14)Gly-Ile-Val-Glu-Gln-Cys-Cys-His-Ser-Ile-Cys-Ser-Trp-Glu-Gln-Leu-Glu-Asn-Tyr-Cys-Asn SEQ ID NO: 81 (des [B1, B2]AB3 des B30): Ala-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe-Tyr- Thr-Pro-LysSEQ ID NO: 82 (des [B1, B2] EB3 des B30):Glu-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe-Tyr- Thr-Pro-LysSEQ ID NO: 83 (des [B1, B2] EB3 EB29)Glu-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe-Tyr- Thr-Pro-Glu-ThrSEQ ID NO: 84 (des [B1, B2] AB3 EB29)Ala-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe-Tyr- Thr-Pro-Glu-ThrSEQ ID NO: 85 (des [B1, B2] EB3 FB17 des B30):Glu-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Phe-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe-Tyr- Thr-Pro-LysSEQ ID NO: 86 (des [B1, B2] EB3 EB17 des B30):Glu-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Glu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe-Tyr- Thr-Pro-LysSEQ ID NO: 87 (des [B1, B2] EB3 NB17 des B30):Glu-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Asn-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe-Tyr- Thr-Pro-LysSEQ ID NO: 88 (des [B1, B2] EB3 FB17 EB29)Glu-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Phe-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe-Tyr- Thr-Pro-Glu-ThrSEQ ID NO: 89 (des [B1-B3] des B30):Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe-Tyr-Thr- Pro-LysSEQ ID NO: 90 (des [B1-B3] QB13 des B30):Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Gln-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe-Tyr-Thr- Pro-LysSEQ ID NO: 91 (des [B1-B3] EB29)Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe-Tyr-Thr- Pro-Glu-ThrSEQ ID NO: 92 (des [B1-B3] QB13 EB29)Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Gln-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe-Tyr-Thr- Pro-Glu-ThrSEQ ID NO: 93 (des [B1-B3] QB13 EB22 des B30):Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Gln-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Glu-Gly-Phe-Phe-Tyr-Thr- Pro-LysSEQ ID NO: 94 (des [B1-B3] QB13 FB17 des B30):Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Gln-Ala-Leu-Tyr-Phe-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe-Tyr-Thr- Pro-LysSEQ ID NO: 95 (des [B1-B3] FB17 des B30):Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Phe-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe-Tyr-Thr- Pro-LysSEQ ID NO: 96 (des [B1-B3] FB17 EB29)Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Phe-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe-Tyr-Thr- Pro-Glu-ThrSEQ ID NO: 97 (des [B1-B3] QB13 FB17 EB29)Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Gln-Ala-Leu-Tyr-Phe-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe-Tyr-Thr- Pro-Glu-ThrSEQ ID NO: 98 (des [B1-B3] EB4 des B30):Glu-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe-Tyr-Thr- Pro-LysSEQ ID NO: 99 (des [B1-B3] AB4 des B30):Ala-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe-Tyr-Thr- Pro-LysSEQ ID NO: 100 (des [B1-B3] EB4 EB29)Glu-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe-Tyr-Thr- Pro-Glu-ThrSEQ ID NO: 101 (des [B1-B3] AB4 EB29)Ala-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe-Tyr-Thr- Pro-Glu-ThrSEQ ID NO: 102 (des [B1-B3] EB4 FB17 des B30):Glu-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Phe-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe-Tyr-Thr- Pro-LysSEQ ID NO: 103 (des [B1-B3] EB4 EB17 des B30):Glu-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Glu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe-Tyr-Thr- Pro-LysSEQ ID NO: 104 (des B1 KB2 AB3 EB17):Lys-Ala-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Glu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe- Tyr-Thr-Pro-Lys-ThrSEQ ID NO: 105 (des B1 KB2 AB3 FB17):Lys-Ala-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Phe-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe- Tyr-Thr-Pro-Lys-ThrSEQ ID NO: 106 (des B1 KB2 EB3 EB17):Lys-Glu-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Glu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe- Tyr-Thr-Pro-Lys-ThrSEQ ID NO: 107 (des B1 KB2 EB3 FB17):Lys-Glu-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Phe-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe- Tyr-Thr-Pro-Lys-ThrSEQ ID NO: 108 (KB2 AB3 EB17):Phe-Lys-Ala-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Glu-Val-Cys-Gly-Glu-Arg-Gly-Phe- Phe-Tyr-Thr-Pro-Lys-ThrSEQ ID NO: 109 (KB2 AB3 FB17):Phe-Lys-Ala-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Phe-Val-Cys-Gly-Glu-Arg-Gly-Phe- Phe-Tyr-Thr-Pro-Lys-ThrSEQ ID NO: 110 (KB2 EB3 EB17):Phe-Lys-Glu-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Glu-Val-Cys-Gly-Glu-Arg-Gly-Phe- Phe-Tyr-Thr-Pro-Lys-ThrSEQ ID NO: 111 (KB2 EB3 FB17):Phe-Lys-Glu-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Phe-Val-Cys-Gly-Glu-Arg-Gly-Phe- Phe-Tyr-Thr-Pro-Lys-Thr

Table 1 below provides a listing of examples of insulin analogues ortheir proinsulin precursors according to the present invention that havebeen synthesized or envisioned. A representative sampling of the insulinanalogues have been tested for biological activity as shown.

TABLE 1 Examples of Insulin Analogs of the Present Invention Maximum T-Structure blood Glucose 0644 DesB1 HA8 EA14 EB29 0674 DesB1 HA8 EA14desB30 0677 DesB1 HA8 EA14 desB30 0679 DesB1 HA8 EA14 EB29 0680 DesB1EA8 EA14 QA17 desB30 17 0681 DesB1 EA8 EA14 QA17 EB29 16 0684 DesB1 EA8EA14 QA17 QB13 desB30 0685 DesB1 EA8 EA14 QA17 QB13 EB29 0688 DesB1 EA8EA14 RA17 desB30 27 0689 DesB1 EA8 EA14 RA17 QB13 EB22 0691 DesB1 EA8EA14 RA17 QB13 EB22 22 0692 DesB1 HA8 EA14 RA17 EB22 desB30 0694 DesB1HA8 EA14 RA17 EB22 desB30 24 0695 DesB1 HA8 EA14 QA17 EB2 desB30 25 0697DesB1 EA8 EA14 QA17 QB13 desB30 27 0701 DesB1 EA8 EA14 QA17 desB30 0702DesB1 EA8 EA14 QA17 EB29 0703 DesB1 HA8 EA14 QA17 desB30 0704 DesB1 HA8EA14 QA17 EB29 0708 DesB1 EA8 EA14 QA17 EB2 desB30 25 0711 DesB1 EA8EA14 QA17 QB13 EB29 Yes 0712 DesB1 HA8 EA14 QA17 EB2 EB29 31 0715 DesB1HA8 EA14 QA17 AB2 desB30 38 0719 DesB1 EA8 EA14 QA17 AB2 desB30 43 0722DesB1 EA8 EA14 RA17 AB2 desB30 25 0724 DesB1 HA8 WA13 desB30 0731 DesB1EA8 EA14 QA17 EB2 EB29 0732 DesB1 EA8 EA14 RA17 EB2 desB30 28 0735 DesB1EA8 EA14 RA17 AB2 desB30 0737 DesB1 HA8 EA14 QA17 AB2 EB29 34 0740 DesB1EA8 EA14 RA17 EB2 desB30 0741 DesB1 EA8 EA14 RA17 desB30 0749 DesB1 HA8LA14 QA17 EB2 desB30 0752 DesB1 EA8 EA14 QA17 AB2 EB29 0757 DesB1 EA8LA14 QA17 EB2 desB30 0759 DesB1 EA8 WA13 EB2 desB30 0760 DesB1 EA8 WA13AB2 desB30 0763 DesB1 EA8 EB2 EB17 desB30 0765 DesB1 EA8 EB2 FB17 desB300769 DesB1 EA8 EB2 NB17 desB30 0773 DesB1 EA8 WA13 EB2 EB29 0779 DesB1EA8 LA14 RA17 EB2 desB30 0781 DesB1 EA8 EA14 RA17 AB2 EB17 0782 DesB1EA8 EA14 RA17 AB2 FB17 0645 des[B1-B2] HA8 EA14 EB29 0675 des[B1-B2] HA8EA14 desB30 0 0682 des[B1-B2] EA8 EA14 QA17 desB30 0683 des[B1-B2] EA8EA14 QA17 EB29 0686 des[B1-B2] EA8 EA14 QA17 QB13 36 0687 des[B1-B2] EA8EA14 QA17 QB13 EB29 17.1 0690 des[B1-B2] EA8 EA14 RA17 QB13 EB22 27.50693 des[B1-B2] HA8 EA14 RA17 EB22 18 0706 des[B1-B2] HA8 EA14 QA17 EB3desB30 36 0707 des[B1-B2] HA8 EA14 QA17 desB30 15 0713 des[B1-B2] EA8EA14 RA17 desB30 0716 des[B1-B2] HA8 EA14 QA17 AB3 desB30 43 0720des[B1-B2] EA8 EA14 QA17 AB3 desB30 0723 des[B1-B2] EA8 EA14 QA17des[B1-B2] 0725 des[B1-B2] EA8 EA14 RA17 EB3 desB30 0728 des[B1-B2] HA8EA14 QA17 EB29 0729 des[B1-B2] HA8 EA14 QA17 EB3 EB29 0734 des[B1-B2]EA8 EA14 RA17 AB3 desB30 0743 des[B1-B2] EA8 EA14 QA17 EB3 EB29 0744des[B1-B2] EA8 EA14 QA17 AB3 EB29 0748 des[B1-B2] HA8 LA14 QA17 EB3desB30 0750 des[B1-B2] HA8 EA14 QA17 AB3 EB29 0758 des[B1-B2] EA8 LA14QA17 K EB3 0761 des[B1-B2] EA8 WA13 EB3 desB30 0762 des[B1-B2] EA8 WA13AB3 des B30 0764 des[B1-B2] EA8 EB3 FB17 desB30 0766 des[B1-B2] HA8 EB3EB17 desB30 0767 des[B1-B2] EA8 EB3 EB17 desB30 0768 des[B1-B2] EA8 LA14EB43 NB17 0774 des[B1-B2] EA8 EB3 FB17 EB29 0780 des[B1-B2] EA8 LA14RA17 EB3 desB30 0312 Des[B1-B3] EA8 2CI-B24 POTEE 0626 Des[B1-B3] EA8EA14 QA17 desB30 Yes 0627 Des[B1-B3] EA8 EA14 QB13 desB30 0628Des[B1-B3] EA8 EA14 QA17 QB13 0629 Des[B1-B3] HA8 EA14 QA17 desB30 0630Des[B1-B3] HA8 EA14 QB13 desB30 0631 Des[B1-B3] HA8 EA14 QA17 QB13 0632Des[B1-B3] EA8 QA17 desB30 0633 Des[B1-B3] EA8 EA14 QA17 EB29 Yes 0634Des[B1-B3] EA8 EA14 QB13 EB29 0635 Des[B1-B3] EA8 EA14 QA17 QB13 EB290636 Des[B1-B3] HA8 EA14 QA17 EB29 18 0637 Des[B1-B3] HA8 EA14 QB13 EB290638 Des[B1-B3] HA8 EA14 QA17 QB13 EB29 0639 Des[B1-B3] EA8 QA17 EB290646 Des[B1-B3] EA8 EA14 RA17 desB30 0647 Des[B1-B3] EA8 EA14 RA17 QB13EB22 0648 Des[B1-B3] EA8 WA13 EA14 desB30 0650 Des[B1-B3] HA8 WA13 EA14desB30 0651 Des[B1-B3] HA8 WA13 EA14 QB13 0653 Des[B1-B3] EA8 EA14 QB13FB17 0654 Des[B1-B3] HA8 EA14 FB17 desB30 0655 Des[B1-B3] HA8 EA14 QB13FB17 0658 Des[B1-B3] EA8 WA13 EA14 EB29 0659 Des[B1-B3] EA8 WA13 EA14QB13 EB29 0660 Des[B1-B3] HA8 WA13 EA14 EB29 0661 Des[B1-B3] HA8 WA13EA14 QB13 EB29 0662 Des[B1-B3] EA8 EA14 FB17 EB29 0663 Des[B1-B3] EA8EA14 QB13 FB17 EB29 0664 Des[B1-B3] HA8 EA14 FB17 EB29 0665 Des[B1-B3]HA8 EA14 QB13 FB17 EB29 0676 Des[B1-B3H EA8 EA14 desB30 0678 Des[B1-B3]HA8 EA14 EB29 0714 Des[B1-B3] HA8 EA14 QA17 EB4 desB30 0717 Des[B1-B3]HA8 EA14 QA17 AB4 desB30 0718 Des[B1-B3] EA8 EA14 QA17 EB4 desB30 0721Des[B1-B3] EA8 EA14 QA17 AB4 desB30 0730 Des[B1-B3] HA8 EA14 QA17 EB4EB29 0733 Des[B1-B3] EA8 EA14 FB17 desB30 0738 Des[B1-B3] EA8 EA14 QA17EB4 EB29 0739 Des[B1-B3] EA8 EA14 QA17 AB4 EB29 0742 Des[B1-B3] HA8 EA14QA17 AB4 EB29 0753 Des[B1-B3] EA8 LA14 QA17 Eb4 desB30 0754 Des[B1-B3]HA8 LA14 QA17 EB4 desB30 0771 Des[B1-B3] EA8 EB4 FB17 desB30 0772Des[B1-B3] HA8 EB4 EB17 desB30 0775 Des[B1-B3] HA8 LA14 QA17 EB4 EB29

Table 2 provides a series of insulin analogues that are synthsized fromplasmids in which the 5′-DNA element encoding the N-terminalpre-sequence of the single-chain precursor polypeptide contains adeletion of the codon 5′ to that encoding Phe^(B1). These plasmidconstructs are designated “desB(minus)1” in the following entries. Thosecontaining “KB2” (Lys^(B2)) enable trypsin digestion to yield a des-(B1,B2) analog whereas those containing “KB3” (Lys^(B3)) enable trypsindigestion to yield a des-(B1-B3) analog.

TABLE 2 desB(minus)1 insulin analogues T-Code Description 0686desB(minus)1 EA8 EA14 QA17 KB2 AB3 EB17 0687 desB(minus)1 EA8 EA14 QA17KB2 AB3 FB17 0688 desB(minus)1 EA8 EA14 RA17 KB2 AB3 EB17 0689desB(minus)1 EA8 EA14 RA17 KB2 AB3 FB17 0690 desB(minus)1 HA8 EA14 QA17KB2 AB3 EB17 0691 desB(minus)1 HA8 EA14 QA17 KB2 AB3 FB17 0692desB(minus)1 HA8 EA14 RA17 KB2 AB3 EB17 0693 desB(minus)1 EA8 EA14 QA17KB2 EB3 EB17 0694 desB(minus)1 EA8 EA14 QA17 KB2 EB3 FB17 0695desB(minus)1 EA8 EA14 RA17 KB2 EB3 EB17 0696 desB(minus)1 EA8 EA14 RA17KB2 EB3 FB17 0697 desB(minus)1 HA8 EA14 QA17 KB2 EB3 EB17 0698desB(minus)1 HA8 EA14 QA17 KB2 EB3 FB17 0699 desB(minus)1 HA8 EA14 RA17KB2 EB3 EB17 0700 desB(minus)1 HA8 EA14 RA17 KB2 EB3 FB17

Biological activity and pharmacodynamics were tested in a subset ofcases in male Sprague-Dawley rats (ca. 300 g) rendered diabetic bystreptozotocin. The receptor-binding affinities of the insulin analogueswere in a subset of cases determined in relation to wild-type humaninsulin (data not shown). Values were observed in the range 5-100%relative to wild-type human insulin in studies of the lectin-purifiedand detergent solubilized insulin receptor (isoforms A and B).Dissociation constants (K_(d)) were determined by fitting to amathematic model as described by Whittaker and Whittaker (2005. J. Biol.Chem. 280: 20932-20936); the model employed non-linear regression withthe assumption of heterologous competition (Wang, 1995, FEBS Lett. 360:111-114).

A subset of insulin analogues were tested for foreshortening of the tailof insulin action following IV bolus injection into the tail vein of aSTZ rat. The dose of the analogue in micrograms per 300-gram rat wasadjusted relative to insulin lispro so that similar nadirs were observed(i.e., the maximal drop in blood-glucose concentration). Although ingeneral more mass of analogue was required than mass of insulin lisproto achieve similar glycemic control, in no case was the difference morethan a factor of five. Six analogues of the present invention wereobserved to exhibit foreshortening of the tail (defined as the area overthe curve and less than the buffer control between minutes 90 and 300relative to the total area over the curve between minutes 0 and 300).These are: des-B1, Glu^(A8), Glu^(A14), Arg^(A17), des-B30-insulin(T-0688), des B1, GlUB2, HisA8, GlU^(A14), Gln^(A17), des-B30-insulin(T-0695), des-[B1, B2], Glu^(B3), His^(A8), Glu^(A14), Gln^(A17),des-B30-insulin-insulin (T-0706), des-B1, Ala^(B2), His^(A8), Glu^(A14),Gln^(A17), des-B30-insulin (T-0715), des-B1, Ala^(B2), Glu^(A8),Glu^(A14), Gln^(A17), des-B30insulin (T-0719), and de s-B1, Ala^(B2),Glu^(B29), His^(A8), Glu^(A14), Gln^(A17)-insulin (T-0737).

Potency of these analogues was evaluated in male diabetic Lewis rats.Experimental analogues, insulin lispro, and diluent only controlsolutions were injected subcutaneously, and the resulting changes inblood glucose (BG) were monitored by serial measurements using aclinical glucometer. Dose response was calculated as maximal BG drop inexcess of the diluent control and plotted. Langmuir isotherm was fittedusing iterative weighting to calculate the best-fit dose response curveand EC50 determined as the interpolated dose required to achieve a drophalf way between fitted maximal and minimal drops. All experimentalanalogues were formulated without zinc. Results are provided in FIG. 3.Each analogue tested had a potency at least 2/3 of that of insulinlispro. Three analogues, displayed a potency equal to or greater thaninsulin lispro, des-B1, Glu^(A8), Glu^(A14), Arg^(A17), des-B30-insulin(T-0688), des [B1, B2], Glu^(B3), His^(A8), Glu^(A14), Gln^(A17),des-B30-insulin-insulin (T-0706), and des-B1, Ala^(B2), Glu^(A8),Glu^(A14), Gln^(A17), des-B30insulin (T-0719).

Thermodynamic Stability. The thermodynamic stabilities of the insulinanalogues were probed by CD-monitored guanidine denaturation in a subsetof cases. The method was as described (Hua, Q. X., et al. J. Biol. Chem.283, 14703-16 (2008)). Briefly, the change in free energies of unfolding(ΔG_(u)) was measured by circular dichroism (CD)-detected guanidinedenaturation at 25° C. at pH 7.4. The results indicate that theseanalogues are each as stable (and in fact more stable) to chemicaldenaturation than is insulin lispro (whose free energy of unfolding(ΔG_(u)) at 25° C. is 2.9±0.1 kcal/mole). Results for selected analoguesare given in Table 3 below. The estimates of ΔG_(u) at 25° C. providedin Table 3 were obtained by application of an analogous two-state modelextrapolated to zero denaturant concentration. Such higher values ofΔG^(u) predict enhanced resistance of the present insulin analogues tochemical degradation under zinc-free conditions than would be observedin studies of wild-type insulin or KP-insulin under the same conditions.

A graphic presentation of change in free energies of unfolding valuesfor a similar comparison of human insulin (HI), insulin lispro (lispro),AspB10-KP insulin (DB10 KP), and EA8 EA14 RA17 des B1 des B30 insulinaccording to the invention is provided in FIG. 4. The change in freeenergies of unfolding (ΔG_(u)) was measured by circular dichroism(CD)-detected guanidine denaturation at 25° C. at pH 7.4. Theexperimental analogues were formulated without zinc. The augmentation instability is at least 1 kcal/mole, achieving a value of ΔG_(u) similaror greater than that of Asp^(B10)-insulin, an analogue known in the artto possess sufficient stability to enable effective zinc-freeformulation. These data demonstrate that a zinc-free insulin analog maybe made as stable as Asp^(B10)-insulin without use of an unnaturalamino-acid substitution and with the native Histidine at position B10.

TABLE 3 Thermodynamic Stabilities of Selected Insulin Analogues ΔGu(kcal/ T-code Structure mol) S.D. Cmid S.D. T-0636 HA8 EA14 QA17des[B1-B3] 4.4 0.1 5.4 0.2 EB29 T-0674 HA8 EA14 desB1 desB30 5 0.2 5.80.3 T-0675 HA8 EA14 des[B1-B2] desB30 4.8 0.2 5.5 0.1 T-0680 EA8 EA14QA17 desB1 desB30 5.1 0.2 5.5 0.2 T-0681 EA8 EA14 QA17 desB1 EB29 4.50.1 5.9 0.1 T-0688 EA8 EA14 RA17 desB1 desB30 5.2 0.1 5.8 0.2 T-0690 EA8EA14 RA17 des[B1-B2] 5 0.1 5.9 0.2 QB13 EB22 desB30 T-0694 HA8 EA14 RA17desB1 EB22 5.3 0.1 6 0.1 desB30

Physical Stability—High Molecular Weight Protein Formation. Thestability of analogues of the claimed invention were also tested forstability by analysis of formation of high molecular weight proteins.Nominal U-100 formulations of several insulin analogues were heatstressed at 45° C. for 7 days and assayed for formation of highmolecular weight proteins (HMWP) by reverse-phase HPLC. FIG. 5 showschanges in percentage of HMWP from day 1 to day 7 for analogues: des-B1,Glu^(A8), Glu^(A14), Arg^(A17), des-B30-insulin (T-0688), des B1,Glu^(B2), His^(A8), Glu^(A14), Gln^(A17), des-B30-insulin (T-0695),des-031, B21, Glu^(B3), His^(A8), Glu^(A14), Gln^(A17),des-B30-insulin-insulin (T-0706), des-B1, Ala^(B2), His^(A8), Glu^(A14),Gln^(A17), des-B30-insulin (T-0715), des-B1, Ala^(B2), Glu^(A8),Glu^(A14), Gln^(A17), des-B30insulin (T-0719), and des-B1, Ala^(B2),Glu^(B29), His^(A8), Glu^(A14), Gln^(A17)-insulin (T-0737). Allexperimental analogues were formulated without zinc. A 2 percent arealoss or greater (for the elution profile for the insulin analogue) wasconsidered a failure. Each of the analogues of the claimed inventionexhibited less than 2 percent area loss, and actually less than 1.5percent area loss. Four of the five analogues tested, T-0695, T-0706,T-0715, T-0719, T-0737, all exhibited less than 1 percent area loss. Inthis way, the insulin analogues tested that were formulated without zincwere closer in behavior to standard Humalog® U100 (insulin lispro; EliLilly) than to insulin lispro reformulated in a zinc-free solution.

Assessment of Fibril Formation. Insulin lispro or embodiments of theinsulin analogues of the claimed invention were made 60 μM inphosphate-buffered saline (pH 7.4) containing 0.1% sodium azide andgently rocked at 37° C. in glass vials in the presence of a liquid/airinterface. Aliquots were taken at regular intervals and frozen for lateranalysis of thioflavin T (ThT) fluorescence. The assay was terminated onvisual appearance of cloudiness. Five analogues are thus tested forprolongation of the lag time prior to onset of ThT-positivefibrillation: des-B1, Glu^(A8), Glu^(A14), Arg^(A17) des-B30-insulin(T-0688), des-B1, Glu^(B2), His^(A8), Glu^(A14), Gln^(A17),des-B30-insulin (T-0695), des-[B1, B2], Glu^(B3), His^(A8), Glu^(A14),Gln^(A17), des-B30-insulin-insulin (T-0706), des-B1, Ala^(B2), His^(A8),Glu^(A14), Gln^(A17), des-B30-insulin (T-0715), and des-B1, Ala^(B2),Glu^(B29), His^(A8), Glu^(A14), Gln^(A17)-insulin (T-0737). Whereas onmultiple testing insulin lispro exhibited lag times of 2-4 days underthese conditions, the lag times of each of the analogues tested wasprolonged by a factor of at least tenfold.

A similar assay assay was also performed. Insulin lispro or embodimentsof the insulin analogues of the claimed invention were formulated to afinal concentration of U10 in phosphate-buffered saline (PBS, pH 7.4)containing 0.1% sodium azide and without zinc and gently rocked at 37°C. in glass vials in the presence of a liquid/air interface. 1 μM thioFlavin T (ThT) was added to each solution and 150 μl was added to eachwell. The plate was incubated at 40° C. with a constant linear shake of1000 cpm. Analysis of thioflavin T (ThT) fluorescence byexcitation/emission wavelengths of 440/480 nm. FIG. 6 is a graphcomparing the fibrillation lag time of these insulin analogues andinsulin lispro. The samples tested were: des-B1, Glu^(A8), Glu^(A14),Arg^(A17), des-B30-insulin (T-0688), des-B1, Glu^(B2), His^(A8),Glu^(A14), Gln^(A17), des-B30-insulin (T-0695), des-[B1, B2], Glu^(B3),His^(A8), Glu^(A14), Gln^(A17), des-B30-insulin-insulin (T-0706),des-B1, Ala^(B2), His^(A8), Glu^(A14), Gln^(A17), des-B30-insulin(T-0715), des-B1, Ala^(B2), Glu^(B29), His^(A8), Glu^(A14),Gln^(A17)-insulin (T-0737) and des-B1, Glu^(A8), Glu^(A14), Gln^(A17),Ala^(B2), des-B30-insulin (T-0719).

IGF-R Binding Affinity. As stated above, reduced binding to themitogenic Type 1 IGF receptor (IGF-R1) would be advantageous. Table 4and FIG. 7 provide the binding affinity of several embodiments of theinsulin analogue of the claimed invention relative to insulin lispro.The samples tested were: des-B1, Glu^(A8), Glu^(A14), Arg^(A17),des-B30-insulin (T-0688), des-B1, Glu^(B2), His^(A8), Glu^(A14),Gln^(A17), des-B30-insulin (T-0695), des-[B1, B2], Glu^(B3), His^(A8),Glu^(A14), Gln^(A17), des-B30-insulin-insulin (T-0706), des-B1,Ala^(B2), His^(A8), Glu^(A14), Gln^(A17), des-B30-insulin (T-0715),des-B1, Ala^(B2), Glu^(B29), His^(A8), Glu^(A14), Gln^(A17)-insulin(T-0737) and des-B1, Glu^(A8), Glu^(A14), Gln^(A17), Ala^(B2),des-B30-insulin (T-0719). Studies employed a FLAG epitope-taggedholoreceptor to human type 1 insulin-like growth factor receptor (hIGFR)bound to 96 well plates coated with anti-FLAG monoclonal antibody.Relative affinity is defined as the ratio of dissociation constants asdetermined by competitive displacement of bound ¹²⁵1 labeled humaninsulin. All experimental analogues were formulated without zinc. Eachembodiment of the claimed invention tested here displayed 26 percent orless of the affinity for IGF-R than insulin lispro. Four analogues,T-0688, T-0706, T-0719, and T-0737, displayed less than 20 percent ofthe affinity of insulin lispro for IGF-R1. One analogue, T-0719,displayed less than 10 percent of the binding affinity of insulin lisprofor IGF-R1

TABLE 4 Relative IGF-R1 Binding Affinity IGF1-R Sequence (Rel. Aff.)Binding T-Code Description ≤HI/KP kD Affinity Lispro Lispro 100% T-0688EA8 EA14 RA17 desB1 desB30 11% 6.72 0.15 T-0695 HA8 EA14 QA17 desB1 EB225% 4.33 0.23 desB30 T-0706 HA8 EA14 QA17 des[B1-B2] EB3 18% 6.09 0.16desB30 T-0715 HA8 EA14 QA17 desB1 AB2 26% 4.16 0.24 desB30 T-0719 EA8EA14 QA17 desB1 AB2 5% 38.04 0.03 desB30 T-0737 HA8 EA14 QA17 desB1 AB216% 12.52 0.08 EB29

Mitogenicity—Cyclin regulation. RT-qPCR assay monitored thetranscription responses of mitogenicity probes stimulated by treatmentof different insulin analogues. Expression regulation of two cyclinsserved as major probes. Cyclin D1 up-regulation and cyclin G2down-regulation correlates to the active cell division cycle(proliferation, which is generally correlated to mitogenicity). A ratioof D1/G2 transcription levels gives a picture of the mitogenic potentialof a compound; a higher ratio means more mitogenic potential. In thisassay, a rat myoblast cell line (L6) with high-expression of insulinreceptor (IR) served as the cell model. All experimental analogues wereformulated without zinc. The samples tested were: des-B1, Glu^(A8),Glu^(A14), Arg^(A17), des-B30-insulin (T-0688), des-B1, Glu^(B2),His^(A8), Glu^(A14), Gln^(A17), des-B30-insulin (T-0695), des [B1, B2],Glu^(B3), His^(A8), Glu^(A14), Gln^(A17), des-B30-insulin-insulin(T-0706), des-B1, Ala^(B2), His^(A8), Glu^(A14), Gln^(A17),des-B30-insulin (T-0715), des-B1, Ala^(B2), Glu^(B29), His^(A8),Glu^(A14), Gln^(A17)-insulin (T-0737) and des-B1, Glu^(A8), Glu^(A14),Gln^(A17) Ala^(B2), des-B30-insulin (T-0719). The mRNA accumulation forCyclin D1 and Cyclin G2, and the ratio of Cyclin D1/Cyclin G2 , withtheir standard deviations, are provided in Table 5. The change in CyclinD1 mRNA accumulation is provided in FIG. 8. Each of the embodiments ofthe claimed invention tested possessed a Cyclin D1/Cyclin G2 ratio lessthan that of both human insulin and insulin lispro.

TABLE 5 Cyclin D1 and Cyclin G Expression T-Code Sequence D1 SD D1 G2 SDG2 Cyclin D1/G2 HI HI 5.5 1.0 0.29 0.04 18.8 DB10 DB10 18.0 1.2 0.130.01 138.8 Lispro Lispro 4.5 0.3 0.36 0.03 12.4 T-0688 EA8 EA14 RA17desB1 desB30 2.6 0.3 0.49 0.07 5.4 T-0695 HA8 EA14 QA17 desB1 EB2 desB304.0 0.8 0.34 0.06 11.8 T-0706 HA8 EA14 QA17 des[B1-B2] EB3 3.3 0.1 0.400.13 8.1 desB30 T-0715 HA8 EA14 QA17 desB1 AB2 desB30 3.1 0.1 0.45 0.036.8 T-0719 EA8 EA14 QA17 desB1 AB2 desB30 3.0 0.3 0.47 0.03 6.5 T-0737HA8 EA14 QA17 desB1 AB2 EB29 3.8 1.9 0.48 0.30 7.9

Since most of these analogues also exhibited a foreshortened tail on IVbolus injection in STZ rats (above), these data together demonstratethat tradeoffs can in principle be circumvented to obtain combinationsof modifications to achieve the desired therapeutic and pharmacologicproperties.

We envision the analogues of the present invention providing a methodfor the treatment of diabetes mellitus or the metabolic syndrome. Theroute of delivery of the insulin analogue is by subcutaneous injectionthrough the use of a syringe or pen device. An insulin analogue of thepresent invention may also contain a foreshortened B-chain due todeletion of residues B1, B1-B2, B1-B3 and/or B30. Insulin analogues ofthe present invention may also contain B chains extended by one- or tworesidues (formal positions B31 and B32), at least one of which is anacidic residue (Aspartic Acid or Glutamic Acid).

A pharmaceutical composition may comprise such insulin analogues in aformulation that specifically excludes zinc ions or other divalent metalions to avoid formation of metal-ion-stabilized insulin analoguehexamers. The pH of the formulation is in the range pH 7.0-8.0; a buffer(typically sodium phosphate or Tris-hydrochloride) may or may not bepresent. In such a formulation, the concentration of the insulinanalogue would typically be in the range 0.6-6.0 mM; concentrations upto 6 mM may be used in vial or pen; the more concentrated formulations(U-200 or higher) may be of particular benefit in patients with markedinsulin resistance. Excipients may include agents intended to accelerateabsorption of the hormone from the subcutaneous depot into thebloodstream (such as sodium EDTA or sodium EGTA, arginine, andnicotinamide), glycerol, glycine, Tris-hydrochloride or other buffers,sodium chloride or other salts, and anti-microbial preservatives such asphenol and/or meta-cresol. Such a pharmaceutical composition may be usedto treat a patient having diabetes mellitus or other medical conditionby administering a physiologically effective amount of the compositionto the patient.

Based upon the foregoing disclosure, it should now be apparent that thetwo-chain insulin analogues provided will carry out the objects setforth hereinabove. Namely, these insulin analogues exhibit biologicalactivity (as defined by the nanomoles of protein monomer required tolower the blood-glucose concentration in a mammal on subcutaneous orintravenous injection) similar to that of wild-type insulin but withsufficient intrinsic stability to enable formulation in the absence ofzinc ions (or other divalent metal ions) at protein concentrations inthe range 0.6-6.0 mM. It is, therefore, to be understood that anyvariations evident fall within the scope of the claimed invention andthus, the selection of specific component elements can be determinedwithout departing from the spirit of the invention herein disclosed anddescribed.

The following literature is cited to demonstrate that the testing andassay methods described herein would be understood by one of ordinaryskill in the art.

Brange J, editor. (1987) Galenics of Insulin: The Physico-chemical andPharmaceutical Aspects of Insulin and Insulin Preparations. Berlin:Springer Berlin Heidelberg.

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What is claimed is:
 1. An insulin analogue comprising a modified A-chainpolypeptide and a modified B-chain polypeptide, wherein the modifiedA-chain polypeptide comprises one or more substitutions relative to thewild-type human insulin A-chain selected from the group consisting of: aHis or Glu substitution at position A8; an Ala, Asp, Glu or Hissubstitution at position A12; an Ala, Glu, His, Phe, Tyr or Trpsubstitution at position A13; an Ala, a Glu, His, Gln or Leusubstitution at position A14; a Gln, Ala, His, Leu, Phe, Tyr or Argsubstitution at positon A17; and an Ala, Gly or Glu substitution atposition A21; and wherein the modified B-chain polypeptide comprises oneor more modifications relative to the wild-type human insulin B-chainselected from the group consisting of: a deletion of the amino acid atposition B 1, a deletion of the amino acids at positions B1 and B2, adeletion of the amino acids at position B1-B3, a deletion of the aminoacid at position B30 or a combination thereof; an Ala or Glusubstitution at position B2; a Glu or Ala substitution at position B3:,an Ala, Gln, His, or Leu substitution at B13; an Ala, Arg, Glu, His,Lys, Phe, Trp, Tyr, or Val substitution at B17; a Glu or Lyssubstitution at B28; a Glu substitution at B29.
 2. The insulin analogueof claim 1, wherein the modified A-chain polypeptide comprises a Glusubstitution at position A14.
 3. The insulin analogue of claim 2,wherein the modified A-chain polypeptide additionally comprises a Glnsubstitution at position A17.
 4. The insulin analogue of claim 2,wherein the modified A-chain polypeptide additionally comprises a Argsubstitution at position A17.
 5. The insulin analogue of claim 3 any oneof claims 3, wherein the modified A-chain polypeptide additionallycomprises a His substitution at position A8.
 6. The insulin analogue ofclaim 2, wherein the modified B-chain polypeptide comprises a deletionof the amino acid at position B1.
 7. The insulin analogue of claim 6,wherein the modified B-chain polypeptide additionally comprises adeletion of the amino acid at position B30.
 8. The insulin analogue ofclaim 6, wherein the modified B-chain polypeptide additionally comprisesan Ala substitution at position B2, and optionally a Glu or Alasubstitution at position B3.
 9. The insulin analogue of claim 6, whereinthe modified B-chain polypeptide additionally comprises a Glusubstitution at position B2, and optionally a Glu or Ala substitution atposition B3.
 10. The insulin analogue of claim 6, wherein the modifiedB-chain polypeptide additionally comprises a deletion of the amino acidat position B2 and additionally comprises a Glu or Ala substitution atposition B3, and optionally a Glu or Ala substitution at position B4.11. (canceled)
 12. The insulin analogue of claim 3, wherein the modifiedA-chain polypeptide additionally comprises a Glu substitution atposition A8.
 13. The insulin analogue of claim 12, wherein the modifiedB-chain polypeptide comprises a deletion of the amino acid at position B1, a deletion of the amino acid at position B30, or both.
 14. (canceled)15. (canceled)
 16. The insulin analogue of claim 13, wherein themodified B-chain polypeptide additionally comprises a deletion of theamino acid at position B2 and additionally comprises a Glu or Alasubstitution at position B3, and optionally a Glu or Ala substitution atposition B4.
 17. The insulin analogue of claim 12, wherein the modifiedB-chain polypeptide comprises a deletion of the amino acid at position B1, additionally comprising a Glu or Ala substitution at position B2 andoptionally a Glu substitution at position B29, and optionally a Glu orAla substitution at position B3.
 18. The insulin analogue of claim 1,wherein the modified A-chain polypeptide comprises the polypeptide ofSEQ ID NO: 42 or SEQ ID NO:
 44. 19. The insulin analogue of claim 18,wherein the modified B-chain polypeptide is a polypeptide selected fromthe group consisting of SEQ ID NOs: 43 and 45-48.
 20. A method oflowering the blood sugar of a patient, the method comprisingadministering a physiologically effective amount of an insulin analogueor a physiologically acceptable salt thereof to the patient, wherein theinsulin analogue comprises a modified A-chain polypeptide and a modifiedB-chain polypeptide, wherein the modified A-chain polypeptide comprisesone or more substitutions relative to the wild-type human insulinA-chain selected from the group consisting of: a His or Glu substitutionat position A8; an Ala, Asp, Glu or His substitution at position A12; anAla, Glu, His, Phe, Tyr or Trp substitution at position A13; an Ala, aGlu, His, Gln or Leu substitution at position A14; a Gln, Ala, His, Leu,Phe, Tyr or Arg substitution at positon A17; and an Ala, Gly or Glusubstitution at position A21; and wherein the modified B-chainpolypeptide comprises one or more modifications relative to thewild-type human insulin B-chain selected from the group consisting of: adeletion of the amino acid at position B 1, a deletion of the aminoacids at positions B1 and B2, a deletion of the amino acids at positionB1-B3, a deletion of the amino acid at position B30 or a combinationthereof; an Ala or Glu substitution at position B2; a Glu or Alasubstitution at position B3; an Ala, Gln, His, or Leu substitution atB13; an Ala, Arg, Glu, His, Lys, Phe, Trp, Tyr, or Val substitution atB17; a Glu or Lys substitution at B28; a Glu substitution at B29. 21.(canceled)
 22. (canceled)
 23. (canceled)
 24. (canceled)
 25. (canceled)26. The insulin analogue of claim 16, additionally comprising one ormore of a substitution selected from the group consisting of a Glusubstitution at position B29 and a Phe substitution at position B17. 27.The insulin analogue of claim 2, additionally comprising a Trpsubstitution at position A13 and a His or Glu substitution at positionA8.
 28. The insulin analogue of claim 1, additionally comprising a Leusubstitution at position A14 and a His or Glu substitution at positionA8.